Using Drosophila insect cells, which express ligands of human NK cell receptors, we have shown that target cell lysis by NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. ICAM-1, which is a ligand of beta2 integrin LFA-1, on insect cells was sufficient to induce polarization of granules, but not degranulation, in NK cells. Conversely, engagement of other activation receptors induced degranulation without specific polarization. Lysis of target cells occurred only when polarization and degranulation were induced by the combined engagement of LFA-1 and other activation receptors. These results have shown that cytotoxicity by resting NK cells is tightly controlled by separate or co-operative signals from different receptors for granule polarization and degranulation. Although a contribution by LFA-1 to granule polarization in T cells has been described, polarization requires co-engagement of the T cell receptor. Furthermore, LFA-1-dependent adhesion of T cells requires inside-out signals, which can be delivered by the TCR, by chemokine receptors, or by other receptors. In contrast, and in support of a unique signaling capacity of LFA-1 in NK cells, binding of NK cells to ICAM-1 is signal-dependent, but independent of inside-out signals from other receptors. The interaction of integrins and their ligands is regulated by changes in both affinity and avidity. The beta2 integrin LFA-1 is essential for the formation of tight conjugates between cytotoxic lymphocytes and their target cells. The aim of this project was to determine how the distribution and the movement of ligands on target cells affect recognition and signaling by receptors on NK cells. We have investigated whether the cytoskeleton of target cells and the movement of the ICAM-1 played a role in NK cell adhesion and granule polarization. Conjugate formation was determined by a two-color flow cytometry assay. Granule polarization was visualized by 3-dimensional confocal imaging of fixed NK-target cell conjugates, which were immunostained for perforin-containing granules. Movement of ICAM-1 was followed live by quantitative imaging, using total internal reflection fluorescence (TIRF) microscopy. Pretreatment of an NK-sensitive target cell line with the actin-depolymerizing drug Latrunculin, but not with the F-actin stabilizing drug Jasplakinolide, abrogated conjugation with primary human NK cells and polarization of cytolytic granules. LFA-1-independent signals for degranulation were much less sensitive to pre-treatment of target cells with Latrunculin. ICAM-1, which is tethered to the actin cytoskeleton, was mostly immobile on the target cells but became highly mobile after Latrunculin treatment. Several inhibitors that did not release ICAM-1 had no effect on LFA-1-dependent NK cell responses. Conversely, a cell line in which ICAM-1 was not tethered and highly mobile at the plasma membrane did not form conjugates with NK cells efficiently. Transfection of ezrin, a molecule known to link the cytoplasmic tail of ICAM-1 to the cytoskeleton, in that cell line resulted in immobilization of ICAM-1 and restored conjugate formation with NK cells. These data imply that tethering of ICAM-1 to the actin cytoskeleton of target cells is required to induce LFA-1-dependent signaling in NK cells. The immobility of ICAM-1 at the surface of target cells appears to be necessary for NK cells to get a grip through LFA-1. As recent studies have reported that integrin binding to extracellular matrix can confer cells the ability to sense forces, our results suggest that ICAM-1 tethering to the cytoskeleton of target cells contributes to LFA-1-dependent mechanotransduction in NK cells.